Aryl sulfonic acid salts of alpha-aminobenzylpenicillins



herein by reference.

United States Patent r 3,180,862 ARYL SULFONIC ACID SALTS 0F- ALPHA-AMINOBENZYLPENICHJLINS Herbert H. Silvestri, De Witt, and David A.Johnson, Fayetteville, N.Y., assiguors to Bristol-Myers Company, NewYork, N.Y., a corporation of Delaware No Drawing. Filed Oct. 29, 1962,Ser. No. 233,943 15 Claims. (Cl. 260-2391) This invention relates to newsynthetic compounds of value as antibacterial agents, as nutritionalsupplements in animal feeds, as agents for the treatment of mastitis incattle, as therapeutic agents in poultry and animals, including man, inthe treatment especially of infectious diseases caused by Gram-positiveand Gram-negative bacteria, and as intermediates in the production ofalphaaminophenylmethyl and alpha-amino-substituted-phenylmethylpenicillins. This invention further relates to a process for theisolation of alpha-aminophenylmethyl andalpha-amino-substituted-phenylmethyl penicillins from impure reactionmixtures containing said penicillins.

Antibacterial agents such as benzylpenicillin have proved highlyeffective in the past in the therapy of infections due to Gram-positivebacteria, but such agents suifer from the serious drawbacks of beingineffective against numerous strains of bacteria, e.g., mostGram-negative bacteria. The compounds of the present invention areparticularly useful in that they possess potent antibacterial activityagainst both Gram-positive and Gram-negative bacteria upon eitherparenteral or oral administration and also exhibit resistance todestruction by acid.

'a-Aminobenzylpenicillin and a amino-substituted-benzylpenicillins areknown in the technical literature, having been described, for example,in United States Patent No. 2,985,648, the disclosure of which isincorporated According to the teachings of that patent, the penicillinsare prepared by reaction of 6* aminopenicillanic acid with an acylatingagent such as the acid chloride, acid bromide, acid anhydride, mixedanhydride, etc. of a derivative of a-aminophenylacetic ora-amino-substituted phenylacetic acid in which the amino group isprotected by a carbobenzoxy or other suitable protecting group. Aftercompletion of the acylation reaction, the protecting group is removedfrom the amino group such as by reaction with hydrogen in the presenceof a catalyst.

The known methods for the preparation of a-aminobenzylpenicillins anda-amino-substituted benzylpenicillins by the acylation of6-aminopenicillanic acid result in the preparation of mixtures whichcontain, in addition to the desired penicillin, unreacted6-aminopenicillanic acid, hydrolyzed acylating agent, and products ofside reactions such as the products of the acylating agent reacted withitself and/or with the desired penicillin. Because these compounds mayhave similar solubility charteristics in various media, it is oftendifficult to isolate the desired penicillin from the other reactionproducts.

Accordingly, it is an object of this invention to provide an improvedprocess for the recovery of a-aminobenzylpenicillin andoc-amino-substituted-benzylpenicillins an acylating derivative ofa-aminophenylacetic or alad amino-substituted-phenylacetic acid. It is afurther object of this invention to provide a new group of aryl sulfonicacid saltsof aminobenzylpenicillin andu-aminosubstituted-benzylpenicillin.

Hereinafter, the term an oa-aminobenylpenicillin is intended to includea-amino-substituted-benzylpenicillins as well as u-aminobenzylpenicillinper se. Similarly, the term an a-arninophenylacetic acid is intended toin clude a-amino-substituted phenylacetic acid as well asa-aminophenylacetic acid per se.

These, and other objects are. achieved bythe practice of this inventionwhich, briefly, comprises providing an aqueous solution containing ana-aminobenzylpenicillin selected from the group consisting of the acidshaving the formula wherein R R and R each represents a member selectedfrom the group consisting of hydrogen, nitro, di(lower)- alkylamino,(lower)a1kanoylamino, (lower)alkanoy1oxy, (lower)alkyl, (includingstraight and branched chain saturated aliphatic groups having from 1 to6 carbon atoms inclusive), (lower)alkoxy, sulfamyl, chloro, iodo, bromo,fluoro, trifluoromethyl, (lower)alkylthio, (lower)- alkylsulfonyl, carbo(lower)alkoxy, benzyl, phenethyl, cycloheptyl, cyclohexyl andcyclopentyl; and their sodium, potassium, calcium, aluminum and ammoniumsalts, their nontoxic substituted ammonium salts with an amine selectedfrom the group consisting of trialkylamines, procaine, dibenzylamine,N-benzylbeta-phenethylamine, 1- ephenamine,N,N'-dibenzylethylenediamine, dehydroabietylamine, N,N bisdehydroabietylethylenediamine, N- (lower)alkylpiperidine, and otheramines which have been used to form salts of benzylpenicillin, as wellas easily hydrolyzed esters or amides which may be converted to the freeacid form by chemical or enzymatic hydrolysis. The aqueous solutioncontaining the u-aminobenylpenicillin of Formula I is contacted witha'water-soluble aryl sulfonic acid or salt thereof having the formula (RSO M wherein M is a radical selected from the group consisting ofhydrogen, ammonium, substituted ammonium, the alkali metals and thealkaline earth metals, wherein x is a whole number equal to the valenceof M and wherein R represents a member selected from the groupconsisting of radicals having the formulae in which R R R and R aremembers selected from the group consisting of hydrogen, alkyl containingfrom 1 to 12 carbon atoms (including straight and branched chaingroups), (lower)alkoxy, (lower)alkylthio, nitro, (lower)alkanoylamino,(lower)alkanoy1oxy, sulfamyl, chloro, iodo, bromo, fluoro,trifluoromethyl, (lower)a1kylsulfonyl, carbo(lower)alkoxy, benzyl,phenethyl, cycloheptyl, cyclohexyl and cyclopentyl. The pH of thesolution is adjusted .to within the range of from about 1.5 to 3.5. Areaction product of said a-aminobenzylpenicillin and said aryl sulfonicacid is thereby formed. This reaction product is then recovered from theaqueous medium.

There is provided, according to the practice of this invention, a memberselected from the group consisting of an acid salt having the formulaFormula. 11

wherein R R R and R have the meaning set forth above. Also includedwithin the scope of this invention are easily hydrolyzed esters oramides which are converted to the free acid form by chemical orenzymatic hydrolysis. Also included within the scope of this inventionare the hydrates of these compounds as well as the anhydrous compounds.

The a-carbon atoms of the acyl group (to which the amine sulfonate saltgroup is attached) is an asymmetric carbon atom and the compounds ofthis invention can therefore exist in two optically active isomericforms (the D- and L-diastereoisomers), as well as in a mixture of thetwo optically active forms; all such isomeric forms of the compounds areincluded within the scope of the present invention.

It should be noted in connection with the foregoing consideration of thediastereoisomers of this invention that many isomers other than the twocaused by the asymmetric carbon of the side chain are possible due tothe presence of asymmetric carbon atoms in the 6- aminopenicillanicnucleus. Such additional isomers, however, are not presently significantsince 6-aminopenicillanic acid, which is the product of fermentationprocesses, is consistently of one configuration; such6-aminopenicillanic acid is presently used in the production of thecompounds of this invention.

The aqueous solution containing an a-aminobenzylpenicillin as defined inFormula I, above, may be obtained by dissolving a crystallinea-arninobenzylpenicillin in water. However, the maximum advantages ofthis invention are attained by using an impure solution containing ana-aminobenzylpenicillin. Typically, the aqueous solution containing ana-aminobenzylpenicillin is obtained by the acylation of6-aminopenicillanic acid with an appropriate acylating agent, such asdescribed in United States Patent No. 2,985,648, or by hydrogenation ofan a-azidobenzylpenicillin. This reaction mixture may contain, inaddition to the desired a-aminobenzylpencillin, unreacted6-aminopenicillanic acid and hydrolysis or side reaction products of theacylating agents. Other compounds, such as2-benzylidene-4-phenyl-3-oxazolin-S-one or its aryl substituted analogs,are also frequently present as impurities. The solution containing thea-aminobenzylpenicillin may also be the mother liquor remaining afterthe recrystallization of an oz-aminobenzylpenicillin.

It is preferred that the solution contain from 25 to 100 mg. of ana-aminobenzylpenicillin per ml. of solution. If necessary, the solutionmay be concentrated by placing the solution under a partial vacuum at atemperature of from 30-40 C. for several minutes.

If the aqueous solution containing an a-aminobenzylpencillin alsocontains organic impurities such as in the reaction mixture obtained byacylating 6-aminopencil-. lanic acid, an organic solvent, preferablymethyl isobutyl ketone, is added to the solution. Other solvents whichmay be used include other (lower)ketones, (lower)aliphatic esters suchas butyl acetate, halogenated (lower)- hydrocarbons such as methylenechloride, aromatic hydrocarbons such as toluene and mixtures thereofwith each other or with methyl isobutyl ketone. Generally, any waterimmiscible solvent may be used. The presence of the solvent facilitatescrystallization and results in a purer product. The volume ratio of theorganic solvent to the aqueous phase is not critical. Thus, for everyvolume of water present, there may also be prescut as much as twovolumes or more of the organic solvent or less than volume of theorganic solvent.

Specific aryl sulfonic acids which may be used in accordance with thepractice of this invention include naphthalene sulfonic acid,p-nonylbenzene sulfonic acid, p-toluene sulfonic acid and p-cymenesulfonic acid. The aryl sulfonic acids which may be used are describedin the prior art and many of them are commercially available. It ispreferred to use a concentrated aqueous solution of the free acidalthough the water-soluble ammonium or substituted ammonium, alkalimetal and alkaline earth metal salts of these acids may also be used.Preferably, from about 1 to about 2 moles of the aryl sulfonic acid areused per mole of the a-aminobenzylpenicillin in the solution.

The aryl sulfonic acid is generally contacted with the aqueous solutioncontaining an a-aminobenzylpenicillin at low temperatures, i.e., attemperatures between about 0-10 C., in order to minimize decompositionof the product and losses of the solution as well as to hasten thecrystallization of the product. The pH of the solution during theformation of the aryl sulfonic acid salt of the a-aminobenzylpenicillinshould be within the range of from about 1.5 to 3.5. The pH of thesolution may be higher than 3.5, for example about 4.5, during theaddition of the aryl sulfonic acid. However, the desired salt will notform untli the pH is within the range of from 1.5 to 3.5; the preferredpH range is 1.5 to 2.0. If the pH is not brought to within the range offrom about 1.5 to 3.5 by the addition of the aryl sulfonic acid, mineralacid such as HCl or H may be added to adjust the pH.

Crystallization of the aryl sulfonic acid salt of theaminobenzylpenicillin may be initiated, if necessary, by seeding. Afterprecipitation of the product is completed, it is recovered by anysuitable means, such as by filtration. The product may then be Washedwith water and/ or an organic solvent such as methyl isobutyl ketone andsubsequently dried. The resultant products are generally recovered ashydrates of the aryl sulfonic acid salts.

In a preferred embodiment of this invention, the hydrate of the arylsulfonic acid salt of the particular aaminobenzylpenicillin is convertedto the anhydrous form. This may be accomplished by slurrying the hydratein a large volume of dry acetone (i.e., about 10 ml. of acetone per gramof hydrate). The amount of the acetone used should be such that therewill be less than 2% by weight of water in the acetone slurry after thehydrate (which may be wet to begin with) is slurried in the acetone. Theslurry is then stirred for about three hours at about 25 to 30 C.,filtered, the filter cake washed with about five volumes of acetone anddried at about 50 C. By this procedure, products of very high purity(i.e. greater than purity) may be obtained. Moreover, the resultantanhydrous products tend to be more heat stable than the correspondinghydrate.

Since the aryl sulfonic acid salts of an a-aminobenzylpenicillinproduced in accordance with the practice of this invention (either thehydrate or anhydrous form) possess valuable anti-bacterial properties,they may be used directly as therapeutic agents. They possess about thesame activity against both Gram-positive and Gram-negative bacteria uponeither parenteral or oral administration as do the correspondinga-aminobenzylpenicillins. In their anhydrous form, these compounds aresoluble in methanol, ethanol, formamide, dimethyl formamide and pyridineand are insoluble in other common solvents. Moreover, they are valuableintermediates for the production of the corresponding purejx-aminobenzylpenicillin. I

An a-aminobenzylpeniciliin aryl sulfonate may be con verted to thecorresponding a-aminobenzylpenicillin by temperature. 'Other alkalinematerials, such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, sodium carbonate, ammonium carbonate, potassium carbonate,etc., may also be used. The adjustment of the pH causes precipitation ofthe ol-aminobenzylpenicillin to commence. The pH of the solution is thenadjusted to about 4.5 to 4.6 by the addition of an acid such as HCl andcooled tocomplete precipitation. After precipitation of the aaminobenzylpenicillin is completed, it may be recovered by filtration.The product may then be washed with water and/or an organic solvent suchas methylisobutyl ketone and dried.

By the practice of the process of this invention, much higher yields ofan a-aminobenzylpenicillin from the reaction mixture in which thea-aminobenzylpenicillin has been prepared by the acylation ofd-aminopenicillanic acid may be obtained than has heretofore beenpossible in a large-scale operation. Moreover, thea-aminobenzylpenicillin is obtained in a higher state of purity than byprevious processes.

The following examples illustrate the best modes contemplated forcarrying out this invention.

Example] A reaction mixture'containing u-aminobenzylpenicillin, formedby the acylation of 1 kg. of 6-aminopenicillanic acid, is concentratedat less than 40 C. to about 15 liters. Methylisobutyl ketone (7.5 l.) isadded to the concentrate which is then chilled to -5 C., adjusted to pH1.8-2.0, agitated about five minutes, the resulting emulsion filteredand the filtrates collected. The filter cake is washed with two 1. ofcold water and then with 2.5 l. of methyl isobutyl ketone and these washportions are combined with the filtrate. The aqueous layer and themethyl isobutyl ketone layer of the filtrate are then separated and theorganic solvent layer is discarded. The aqueous layer is adjusted to pH4-5 with triethylamine, maintained at (3-10 C. and mixed with 7.5 l. ofmethyl isobutyl ketone. To the resulting mixture there is added withvigorous agitation 3 l. of an aqueous solution containing 1.35 kg. offimaphthalene sulfonic acid. During addition of the ,B-naphthalenesulfonic acid, the reaction mixture is not allowed to exceed 10 C. andthe pH is maintained above 1.5 by intermittent addition oftriethylamineas required. Following the addition of the acid solution, the pH of thereaction mixture is adjusted to 1.6-2.0, seeded, and agitated for 2 to 4hours at 0 to 5 C. and at a pH of 1.6 to 2.0 whereupona-aminobenzylpenicillin-B-naphthalene sulfonic acid salt precipitates.After precipitation of the product is completed, the reaction mixture isfiltered and the collected product washed twice with 2.5 l. of coldwater and with three successive washes of 2.51. of methyl isobutylketone. The collected product, the monohydrate of fl-naphthalenesulfonic acid salt of tx-aminobenzylpenicillin is dried at 50 C; and

found by bioassay to have 600 mcg. u-aminobenzylpenicillin activity/mg.The theoretical activity of a-aminobenzylpenicilline -naphthalenesulfonic acid salt monohydrate is 606 mcg. a-aminobenzylpenicillin/mg.The product inhibits Staph. aureus Smith at concentrations of 0.001% byWeight.

Example 2 A two hundred gram portion ofa-aminobenzylpenicillin-B-naphthalene sulfonic acid salt (prepared as'described in Example 1) is added with agitation to 1500 ml. of water atroom temperature. Triethylamine (54 ml.)

procedure described in Example 1 above.

. 6 is added to the resulting suspension over a period of severalminutes. Four successive 200 gm. portions of the salt are added to thesuspension, each portion being followed by the addition of 54 ml.triethylamine and by agitation. Some pure a-aminobenzylpenicillincrystallizes out of the solution following each addition. The resultingslurry is agitated at room temperature (25-30 C.) at a pH of 6.7-7.5 forabout one hour. Subsequently, the slurry is chilled and, while chilling,the pH is slowly adjusted over about a 10-30 minute period to about 4.5to 4.6 by the addition of about ml. 6 N hydrochloric acid. After the pHis adjusted, the slurryis agitated at 0 to 5 C. for about two hours andthen filtered. The mother liquors are removed from the collected solidswhich are then washed with two 200 ml. portions of ice water and thenwith 1000 ml. of cold methyl isobutyl ketone. After Washing, thecollected product, a-aminobenzylpenicillin,

is dried at C. and found to weigh about 415 gm. and to assay 1000 rncg.ot-aminobenzylpenicillin/mg. (100% of theoretical activity; 70% oftheoretical yield).

Thecombined mother liquors and wash are chilled, acidified to pH 1.6with 20% sulfuric acid, and main tained at from 0-5 C. for two hours andthe a-aminobenzylpenicillin is recovered therefrom in the form of itsB-naphthalene sulfonic acid salt in accordance with the The salt sorecovered is then treated in accordance with the process of this exampleas described above. The over-all yield of ct-aminobenzylpenicillin fromthe B-naphthalene sulfonic acid salt thereof is about 85% of theoreticalyield.

Example 3 There is obtained 1.9 g. of a compound which is determined byinfra-red analysis to be the fi-naphthalene sulfonic acid salt ofwaminobenzylpenicillin. 1

Example 4 wAminobenzylpenicillin (2.0 g.) is dissolved in 50 ml.

of water by adjusting the pH of the solution to 2.0. There are addedanother 5 0 ml. of water followed by the drop- Wise addition of asolution of 1.36 g. of sodium [-I-naphthalene sulfonate in 15 ml. ofwater. Methyl isobutyl ketone is added (30 ml.) and the solution isstirred. Crystals are thereby formed which are recovered by filtration,washed and dried. The product (2.4 g.) is the fl-naphthalene sulfonicacid salt of a-aminobenzylpenicillin.

Example 5 To 50 ml. of the acidified mother liquors (pH 2.0) from whichu-aminobenzylpenicillin has been recrystallized, there are added 1136 g.of sodium naphthalene sulfonic acid'in 15 ml. of water and 25 ml. ofmethyl isobutyl ketone. An oil is formed followed by crystallization.The product is recovered by filtration, washed with water and methylisobutyl ketone and dried under vacuum. The product (1.7 g.) is theB-naphthalene sulfonic acid salt of a-aminobenzylpenicillin.

Example 6 ketone is added to the aqueous phase and then a solutionconsisting of fl-naphthalene sulfonic acid (16.0 g.) and concentrated H80 (2.3 g.) in 50 ml. of water is added dropwise to the mixture whilethe pH of the mixture is maintained at from 1.8 to 2.0 by the periodicaddition of NH OH. After /3 f the [El-naphthalene sulfonic acid-H 50solution is added, the mixture is seeded witha-aminobenzylpenicillin-[3-naphthalene sulfonic acid and a crystallineproduct commences to form. The remainder of the B-naphthalene sulfonicacid-H 50 solution is added over a 20-minute period. The mixture isallowed to stand at room temperature for 30 minutes during which timecrystallization of the product proceeds. The product is then recoveredby filtration and washed with water and methyl isobutyl ketone. Thisproduct (10.4 g.) is the monohydrate ofa-aminobenzylpenicillin-B-naphthalene sulfonic acid salt. The dampfilter cake, which contains less than 20% by weight of water, isslurried in 104 ml. of dry acetone. The slurry is stirred for threehours at 25-30 C., filtered, the filter cake is washed with 52 ml. ofdry acetone and dried at 50 C. The product, anhydrousa-aminobenzylpenicillin ,B-naphthalene sulfonate, has an activity of 618mcg. a-aminobenzylpenicillin/mg. which corresponds to a purity of 99%.It is very heat stable and shows no potency loss after storage for fourweeks at 70 C. Exposure to laboratory atmosphere for three days resultsin no moisture pickup. The product inhibits Staph. aureus Smith at aconcentration of 0.001% by weight.

Example 7 An aqueous reaction mixture (300 ml.) which has been formed bythe acylation of 6-aminopenicillanic acid and which containsa-amino-meta-chlorobenzylpenicillin in admixture with various impuritiesis filtered. The filtrate (pH 7.1) is adjusted to pH 5.0 and ammoniumB-naphthalene sulfonate (2.9 g.) is added with rapid stirring. The pH ofthe clear, yellow solution is slowly lowered to 2.0 with concentrated HSO causing a quantity of oil and gum to separate. The mixture is thenlayered with 60 ml. of methyl isobutyl ketone and the mixture is stirredvigorously with occasional scratching. In a short time, theB-naphthalene sulfonate of a-amino-metachlorobenzylpenicillincrystallizes from the solution. The crystalline product is collected byfiltration and, while still slightly damp, is suspended in 75 ml. ofwater. The pH of the suspension is adjusted to 7.5 by the addition oftriethylamine. At first, most of the solid material dissolves. Then, acrystalline material begins to separate. The pH of the mixture isadjusted to 4.3 by the addition of 6 N HCl. After /2 hour, theprecipitated crystalline material is collected by filtration, driedovernight in vacuo over P 0 The product (0.90 g.) is determined byinfra-red analysis to be substantially purea-aminometa-chiorobenzylpenicillin.

Example 8 a-Aminobenzylpenicillin (2 g.) is dissolved in 70 ml. of waterand the pH of the solution is adjusted to 7.8 by the addition of NH OH.A clear solution results. A solution comprising 2.50 g. ofp-nonylbenzene sulfonic acid in 10 ml. of water, the pH of which hasbeen adjusted to 2.5 by the addition of 20% H 80 is added to thesolution of a-aminobenzylpenicillin. There is then added 20 ml. ofmethyl isobutyl ketone and the pH of the mixture is adjusted to 1.5.Crystal formation cornmences at this point. The mixture is chilled forone hour, filtered, washed with water and 4 ml. of methyl isobutylketone and dried at 50 C. The product (3.45 g.) is the monohydrate ofthe p-nonylbenzene sulfonic acid salt of a-aminobenzylpenicillin. It hasan activity of 535 meg/mg. and is determined to contain the B-lactamstructure by infra-red analysis. The product inhibits Staph. azu'eusSmith at concentrations of 0.001% by weight.

8 Example 9 a-Aminobenzylpenicillin (2.0 g.) is dissolved in 40 ml. ofwater having a pH of 7.9. To the clear solution there is added asolution (pH 2.5) of 1.93 g. of sodium p-cymene sulfonate in 10 ml. ofwater. There are then added to the mixture 20 ml. of methyl isobutylketone and the pH of the mixture is adjusted to 1.5. The mixture ischilled, whereupon crystallization occurs. The precipitated product isrecovered by filtration, washed with water and Skellysolve B and dried.The product (3.0 g.) is determined by infra-red and elemental analysisto be the trihydrate of p-cymene sulfonic acid salt ofa-aminobenzylpenicillin and to have an activity of 610 mcg./mg. Theproduct inhibits Staph. aureus Smith at concentrations of 0.001% byweight.

Example 10 a-Arninobenzylpenicillin (2.0 g.) is dissolved in 40 ml. ofwater having a pH of 7.7. To this clear solution there is added 1.6 g.of sodium toluene sulfonate dissolved in 10 ml. of water. The pH of theclear solution is adjusted to 2.0 and 10 ml. of methyl isobutyl ketoneis added. The pH of the mixture is adjusted to 1.5, and the mixture ischilled for one hour whereupon crystallization occurs. The precipitatedproduct is recovered by filtration, washed with water and Skellysolve Band dried. The product (1.6 g.) is found to have an activity of 690meg/mg. and is determined to be a a-aminobenzylpenicillin toluenesulfonate. The product inhibits Staph. aureus Smith at concentrations of0.001% by weight.

Example 11 a-Carbobenzyloxyaminophenylacetic acid (0.1 mole), which isobtained by the reaction of equivalent quantities of a-aminophenylaceticacid and benzylchlorocarbonate in aqueous sodium hydroxide, dissolved indry acetone, is mixed with triethylamine (0.2 mole), stirred and cooledto approximately 5 C. To this there is added dropwise with continuedcooling and stirring a solution of ethyl chlorocarbonate (0.1 mole).After approximately ten minutes, the acylating mixture is cooled toabout -5 C. and then is slowly added to a stirred ice-cold mixture of6-aminopenicillanic acid (0.1 mole), 3% sodium bicarbonate solution (0.1mole) and acetone. This reaction mixture is allowed to attain roomtemperature, stirred for an additional thirty minutes at thistemperature and then is extracted with ether. The extracted aqueoussolution is covered with butanol and the pH adjusted to 2 by theaddition of N HCl. The acidified aqueous phase is extracted withbutanol, the pH of the aqueous phase being adjusted to pH 2 each time.The combined butanol solutions which contain the free acid,a-carbobenzyloxyaminobenzylpenicillin, are washed with water, and arethen shaken with water to which sufiicient 3% sodium bicarbonate hasbeen added to bring the aqueous phase to pH 7. This process of washingand shaking is repeated with fresh water and bicarbonate solution. Thecombined aqueous solutions are washed with ether and then are evaporatedunder reduced pressure and low temperature. The product, the sodium saltof a-carbobenzyloxyaminobenzylpenicillin, is obtained as a yellow solidin a yield of 65 percent.

A suspension of palladium in barium carbonate (3.7 g. of 30%) in water(20 ml.) is shaken in an atmosphere of hydrogen at room temperature. Thecatalyst is then filtered and washed well with water, care being takenthat it does not become dry. A solution of the sodium salt ofa-carbobenzyloxyaminobenzylpenicillin (4 g.) in water (20 ml.) is addedto the pretreated catalyst and the suspension is shaken in an atmosphereof hydrogen at room temperature and pressure for one hour. The catalystis then filtered ott, washed well with water, and the combined filtrateand washings adjusted to pH 7 disease with N hydrochloric acid. Thesolution is then concentrated to about 40 ml. by heating at about 40 C.To the concentrated solution, there is added with agitation a solutionof 1.4 g. of ,B-naphthalene sulfonic acid in 15 ml. of water while thetemperature of the concentrate is maintained below 10 C. and the pH ismaintained above 1.5 by intermittent addition of triethylamine.Following' the addition of the acid solution, the pH of the reactionmixture is adjusted to.1.7, seeded by the addition of fi-naphthalenesulfonic acid salt of u-aminobenzylpenicillin and agitated for threehours at 3- C. and at pH 1.7. The monohydrate of a-aminobenzylpenicillin[i-naphthalene sulfonate which precipitates is collected by filtrationand washed twice With cold water, and with three successive washes ofmethyl isobutyl ketone. It is found to contain the B-lactam structure asshown by infra-red analysis, inhibits Staph. aureus at a concentrationof 0.012 mcg./ml., and upon intra-muscular injection in mice, exhibitsversus Staph. (ZLlrfllS Smith, Salm. typhimurium and Klebs. pnewmoniae,a CD ot G.05 mg./kg., 35 mg./kg. and 62 mg./kg., respectively.

Example 2 A portion of the damp filter cake comprising the monohydrateof a-aminobenzylpenicillin fl-naphthalene sulfonate obtained in Example11 (1.0 g.) which contains less than 20% by weight of Water is slurriedin 10 ml. of dry acetone. The slurry is stirred for two hours at 30' C.,filtered, the filter cake washed with ml. of dry acetone anddried atabout 50 C. The product, anhydrous oz' aminobenzylpenicillin[El-naphthalene sulfonate, has an activity of 618 mcg./ mg. It is veryheat stable and'shows no potency loss after being stored for four weeksat 70 C.

Example 13 -The product obtained in Example 12 (anhydrousaamino-benzylpeniciilin fi-naphthalene sulfonate) is sus-' pended inabout 50 m1. of Water. The pH of the suspen sion is adjusted to 7.5 bythe addition of triethylainine whereupon a crystalline material beginsto separate. The pH of the mixture is then adjusted to about 4.5 by theaddition of 6 N HCl. The mixture is allowed to crystallize for about /2hour and then the precipitated crystalline material is collected byfiltration and dried. The product is determined by infra-red analysis tobe subtantially pure a-aminobenzylpenicillin.

Example 14 When in the procedure of Example 11 thea-carbobenzyloxyaminophenylacetic acid is replaced by 0.1 mole ofExample 15 Replacement of the a-aminobenzylpenicillin S-naphthalenesulfonate in Example 13 with Example 16 7 When in the procedure ofExample 11, the fi-naphthalene sulfonic acid is replaced by anequivalent molar amount of p-Chlorobenzene sulfonic acid,

m-Nitrobenzene sulfonic acid,

6,7-dihydroxy-3-naphtha1ene sulfonic acid,

4-acetamidobenzene sulfonic acid,

2,4-dimethylbenzene sulfonic acid,

p-Methoxybenzene sulfonic acid, and

p-Acetoxybenzene sulfonic acid, respectively, the following compounds orhydrates thereof are produced:

u-Arninobenzylpenicillin-p-chlorobenzene sulfonate,

at-Aminobenzylpenicillin-m-nitrobenzene sulfonate,

a-Aminobenzylpenicillin-6,7-dihydroxy-2-naphthalene sulfonate,

a-Aminobenzylpenicillin-4-acetamidobenzene sulfonate,

a-Aminobenzylpenicillin-2,4-dimethylbenzene sulfonate,

m-Arninobenzylpenicillin-p-methoxybenzene sulfonate, and

ct-Aminobenzylpenicillin-p-acetoxybenzene sulfonate.

' We claim:

1. A member selected from the group consisting of wherein R R and R eachrepresents a member selected from the group consisting of hydrogen,nitro, di(lower) alkylamino, (lower)alkanoylamino, (lower)alkanoyloxy,(lower)'talkyl, (1ower)all oxy, sulfamyl, chloro, iodo, bromo, fiuoro,trifiuoromethyl; and their sodium, potassium, calcium, aluminum andammonium salts, their nontoxic salts with an amine selected from thegroup consisting of tri(lower) alkylamines, procaine, dibenzylamine,N-benzyl-beta-phenethylamine, l-ephenamine, N,N-dibenzylethylenediamine,dehydroabietylamine, N,N-bisdehydroabietylethylenediamine andN-(lower)alkylpiperidine, and wherein R represents a member selectedfrom the group consisting of radicals having the formulae and B U R inwhich R R R and R are members selected from the group consisting ofhydrogen, alkyl containing from 1 to 12 carbon atoms, (lower)alkoxy,nitro, (lower alkanoylamino, chloro, iodo and bromo.

2. a-Aminobenzylpenicillin p-naphthalene sulfonate. 3.a-Amino-m-chlorobenzylpenicillin B-naphthalene sulfonlate.

4. a-Aminobenzylpenicillin p-nonylbenzene sulfonate. 5.a-Aminobenzylpenicillin p-cymene sulfonate. 6. a-Aminobenzylpenicillinp-toluene sulfonate. 7. A process which comprises: providing an aqueoussolution containing an a-aminobenzyl-penicillin selected from the groupconsisting of the acids having the formula wherein R R and R eachrepresents a member selected from the group consisting of hydrogen,nitro, di(lower)alkylamino, (lower)alkanoylarnino, (lower)alkanoyloxy,(lower)alkyl, (lower) alkoxy, sulfamyl, chloro, iodo, brorno, fluoro,trifluoromethyl; and their sodium, potassium, calcium, aluminum andammonium salts, their nontoxic salts with an amine selected from thegroup consisting of tri(lower) alkylamines, procaine, dibenzylamine,N-benzylbetaphenethylamine, l-ephenamine, N,N-dibenzylethylenediamine,dehydroabietylamine, N,N-bis-dehydroabietylethylenediamine, andN-(lower)alkylpiperidine;

contacting said solution at about C. to 50 C. with a water-soluble arylsulfonic acid having the formula (R"-SO M wherein M is a radicalselected from the group consisting of hydrogen, ammonium, the alkalimetals and the alkaline earth metals, wherein x is a whole number equalto the valence of M and wherein R represents a member selected from thegroup consisting of radicals having the formulae I Q and w R5 0 in whichR R R and R are members selected from the group consisting of hydrogen,alkyl containing from 1 to 12 carbon atoms, (lower)alkoxy, nitro,(lower)alkanoylamino, chloro, iodo, and bro mo;

adjusting the pH of the reaction mixture to within the range of fromabout 1.5 to 3.5 whereby a reaction product of saida-aminobenzylpenicillin and said aryl sulfonic acid is formed;

and recovering said reaction product.

8. The process of claim 7 wherein said a-aminobenzylpenicillin isa-aminobenzylpenicillin and wherein said aryl sulfonic acid isfl-naphthalene sulfonic acid.

9. A process for the recovery of an a-aminobenzylpenicillin from animpure solution thereof which comprises:

providing an impure aqueous solution containing anot-aminobenzylpenicillin selected from the group consisting of the acidshaving the formula wherein R R and R each represents a member selectedfrom the group consisting of hydrogen, nitro, di(lower)alkylamino,(lower)alkanoylamino, (lower)alkanoyloxy, (lower)alkyl, (lower)alkoxy,sulfamyl, chloro, iodo, bromo, fluoro, trifiuoromethyl, and their watersoluble salts;

adding to said aqueous solution an organic water immiscible solvent toform a mixture;

contacting said mixture at about 0 C. to C. with a water-soluble arylsulfonic acid selected from the group consisting of p-naphthalenesulfonic acid, pnonylbenzene sulfonic acid, p-cymene sulfonic acid,p-toluene sulfonic acid and their ammonium and alkali metal salts;

adjusting the pH of said mixture to within the range of from about 1.5to 3.5 whereby a crystalline reaction product of said ana-aminobenzylpenicillin and said aryl sulfonic acid is formed;

recovering said crystalline reaction product; suspending said reactionproduct in water, adjusting the pH of said suspension to within therange of from about 6.0 to 8.0 and recovering the resultant ana-aminobenzylpencillin.

10. The process of claim 9 wherein said an a-aminobenzylpenicillin isa-aminobenzylpenicillin and wherein said aryl sulfonic acid isfi-naphthalene sulfonic acid.

11. The process of claim 10 wherein said recovered crystalline reactionproduct of said an a-aminobenzylpenicillin and said arylsulfonic acid isslurried in acetone and subsequently recovered therefrom.

12. The process of claim 9 wherein said aryl sulfonic acid is contactedwith said mixture at a temperature between about 0 and 10 C. and whereinthe pH of said mixture is from 1.5 to 2.0.

13. The process of claim 9 wherein said recovered reaction product issuspended in water, the pH of said suspension is adjusted to within therange of 6.7 to 7.2 and subsequently to about 4.5 to 4.6.

14. A process for the production of the anhydrous 13- naphthalenesulfonic acid salt of an a-aminobenzylpenicillin which comprises:

providing an aqueous solution containing an a-aminoenbzylpenicillinselected from the group consisting of the acids having the formulawherein R R and R each represents a member selected from the groupconsisting of hydrogen, nitro, di(lower)alkylamino,(lower)alkanoylamino, (lower) alkanoyloxy, (lower) alkyl, (lower)alkoxy, sulfamyl, chloro, iodo, bromo, fluoro, trifiuoromethyl, andtheir sodium, potassium, calcium, aluminum and ammonium salts, theirnontoxic substituted ammonium salts with an amine selected from thegroup consisting of tri(lower)alkylamines, procaine, dibenzylamine, Nbenzyl beta phenethylamine, 1- ephenamine, N,N-dibenzylethylenediamine,dehydroabietylamine, N,N' bis-dehydroabietyl-ethylenediamine andN-(lower)alkylpiperidine;

contacting said solution at about 0 C. to 50 C. with fi-naphthalenesulfonic acid;

adjusting the pH of the reaction mixture to within the range of fromabout 1.5 to 3.5 whereby the mono- 3,180,862 13 14 hydrate of said anu-aminobenzylpenicillin (i-naph- References Cited by the Applicantthalene SUIfOIlEliZC iS fOI'l'llGdI ST P recovering said monohydrate,slurrying said monohydrate in acetone and subsequently recovering from2,223,935 12/40 Danlels at said slurry the anhydrous ii-naphthalenesulfonic acid 5 2,876,236 3/59 Szabo et salt of saida-aminohenzyipenicillin. 2,985,648 5/61 Doyle at 15. The process ofclaim 14 wherein said an u-amin0 OTHER REFERENCES benzylpenicflliniswaminobenzylpenicimn' Aldrich Chemical Company, Inc., Catalog No. 11,

- pages 338-346 (1963), References cued by the Exammer 10 DistillationProducts Industries, Eastman Organic UNITED STATES PATENTS ChemicalsClassified by Functional Groups No. 2F,, 2,223,935 12/40 Daniels et al.260-501 p g 5345 2,876,236 3/59 Szabo et a1. 260-2391 2,935,648 5/61Doyle et 1 1 NICHOLAS S. RIZZO, Primary Exammer.

3,157,640 11/64 Johnson et a1. 260-239.1 15

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,180,862 April 27, 1965 Herbert H. Silvestri et 211.

It is hereby certified that error appears in the above numbered patentrequiring correction and that the said Letters Patent should read ascorrected below.

Column 2, lines 16 to 21, column 11, lines 25 to 30, and column 12,lines 3 to 8, the formula, each occurrence, should appear as shown belowinstead of as in the patent:

ca H-:LNH H-C c 3 i |\CH3 2 NHZ Q N cacooa column 2, line 35, for"Nbenzylbeta-phenethylamine" read N-benzyl-beta-phenethylamine column 4,line 34, for "untli" read until column 10, lines 58 to 62, the formulashould appear as shown below instead of as in the patent:

0 s ?a!: m1 ca \C CH3 -R4SO3H 2 N11 I CH3 R 3 0 c N cncoon Signed andsealed this 5th day of October 1965.

SEAL) ttest I RNEST W. SWIlJER EDWARD J. BRENNER ttestlng OfficerCommissioner of Patents

1. A MEMBER SELECTED FROM THE GROUP CONSISTING OF
 7. A PROCESS WHICHCOMPRISES: PROVIDING AN AQUEOUS SOLUTION CONTAINING ANA-AMINOBENZYL-PENICILLIN SELECTED FROM THE GROUP CONSISTING OF THE ACIDSHAVING THE FORMULA